The long term goal of this project is to enhance our understanding of the NF-?B inhibitor, I?B?, and its regulatory roles in T cell biology. Followin recognition of cognate antigen through the T cell receptor, T cell activation of the NF-?B signaling pathway results in the degradation of I?B? and release of active NF-?B dimers from the cytoplasm to the nucleus. Upon nuclear localization, NF-?B binds to DNA and stimulates transcription of target genes, including I?B? (Nfkbia). Newly synthesized I?B? terminates signaling by removing NF-?B dimers from DNA and exporting the entire inactive complex out of the nucleus, and restoring an inactive pool of NF-?B in the cytoplasm. This process is promoted by a nuclear export sequence (NES) present on I?B?. It is proposed that the ability of I?B? to dynamically localize between the nucleus and cytoplasm plays a key regulatory role in controlling NF-?B signaling. Our group has generated a unique transgenic mouse model, NfkbiaNES/NES, with a germline mutation in the NES of I?B?, to understand the biological impacts of modulating NF-?B signaling in vivo. Surprisingly, we have found enhancements in NfkbiaNES/NES CD4+ T cell proliferation in vitro and T follicular helper (Tfh) cell differentiationin vivo during an acute (LCMV) viral infection. We hypothesize that this can be explained by a downregulation of a critical negative regulator of NF-?B signaling, microRNA mir-146a. A thorough understanding of how the nuclear export of I?B? regulates NF-?B signaling will be critical for understanding T cell function and differentiation in a pathogenic setting. The first am of this proposal will be to determine whether NF-?B activation functionally regulates NfkbiaNES/NES CD4+ T cell function in a mir-146a dependent manner. Assays to determine activation of specific NF-?B family members will be used to address this aim. In addition, genetic manipulation of WT and NfkbiaNES/NES CD4+ T cells will be utilized to determine the requirement for mir-146a in the hyperproliferative, Tfh-like phenotype we observe. The second aim of this proposal is directed at defining the physiological relevance of I?B? nuclear export in CD4+ T cells during an acute LCMV infection model. Our results have revealed an enhancement in differentiation of CD4+ T cells to Tfh cells. Tfh cells act as key cellular regulators of antibody production. This will be the first mechanistic in vivo study testing whether regulation of I?B? nuclear export function in NF-?B signaling differentially regulates T cell differentiation in a pathogenic setting. In line with the mission of NIAID to, expand the breadth and depth of knowledge in all areas of infectious, immunologic, and allergic diseases, this proposal will provide new insights into the mechanisms by which NF-?B signaling regulates immunological function that can aid in the design of antiviral therapies.